Two-colour flow-cytometric analysis of pulmonary alveolar macrophages from smokers.
The study of alveolar macrophages (AM) from smokers by flow cytometry (FCM) has been limited by strong autofluorescence and the lack of reliable markers to identify macrophages. Crystal violet quenching was reported to be effective in reducing autofluorescence of AM. CD68 is a marker for macrophages in immunohistochemistry, but has been less useful in FCM because of poor surface expression. This study evaluated the effectiveness of a method for two-colour FCM analysis of AM combined with membrane permeabilization and crystal violet quenching. Bronchoalveolar lavage cells, fixed in 4% paraformaldehyde and permeabilized using 0.5% Triton X100, were incubated with fluorescent-labelled antibodies for 30 min and quenched with a saturated crystal violet solution. Two-colour FCM was then performed using forward/side scatter gating to select AM. Autofluorescence at 525 nm (fluorescein isothiocyanate) and 575 nm (phycoerythrin) markedly decreased after quenching. After permeabilization, 97.1+/-2.8% of the gated cells were CD68+, while 53.9+/-18.6% of the AM were positive without permeabilization. CD68+ cells were sorted and proved to be AM morphologically. Analysis of CD71 (transferrin receptor) expression by FCM correlated with immunocytochemistry (r=0.77, p<0.05). The permeabilization/quenching technique, therefore, represents a satisfactory means to evaluate alveolar macrophages by flow cytometry. (+info)
Genetically modified Vibrio harveyi strains as potential bioindicators of mutagenic pollution of marine environments.
For biodetection of mutagenic pollution of marine environments, an organism naturally occurring in these habitats should be used. We found that marine bacterium Vibrio harveyi may be an appropriate bioindicator of mutagenic pollution. For positive selection of mutants, we developed a simple method for isolation of V. harveyi mutants resistant to neomycin. We constructed genetically modified V. harveyi strains that produce significantly more neomycin-resistant mutants upon treatment with low concentrations of mutagens than the wild-type counterpart. The sensitivity of the mutagenicity test with the V. harveyi strains is at least comparable to (if not higher than) that of the commonly used Ames test, which uses Salmonella enterica serovar Typhimurium strains. Therefore, we consider that the V. harveyi strains described in this report could be used as potential bioindicators of mutagenic pollution of marine environments. (+info)
Gram stain of bronchoalveolar lavage fluid in the early diagnosis of ventilator-associated pneumonia.
To assess the usefulness of the Gram stain in the early diagnosis of ventilator-associated pneumonia (VAP), we performed 146 protected specimen brushings (PSB) and bronchoalveolar lavages (BAL) in 118 patients suspected of having nosocomial pneumonia. Gram stain and counts of infected cells were performed in all samples from BAL fluid. A final diagnosis of pneumonia was established in 51 patients and there was no infection in 95 cases. A threshold of 2% of infected cells was used to distinguish between VAP and the group without VAP (sensitivity 86.3%, specificity 78.9%, positive predictive value 68.7% and negative predictive value 91.4%); there was good agreement with the final diagnosis (kappa statistic 0.616; concordance 81.5%). Regarding detection of bacteria using the Gram stain, we found a sensitivity of 90.2%, specificity 73.7%, positive predictive value 64.8% and negative predictive value 93.3%; there was moderate agreement with the final diagnosis (kappa statistic 0.586; concordance 79.4%). In the VAP group, we analysed the degree of qualitative agreement between Gram stain and PSB quantitative cultures: the correlation was complete in 51% (26 of 51 VAP), partial in 39.2% (20 of 51 VAP) and there was no correlation in 9.8% (five of 51 VAP). We conclude that the Gram stain is useful for rapid diagnosis of VAP but unreliable for early adaptation of empiric therapy. (+info)
Gliotoxin-induced cytotoxicity proceeds via apoptosis and is mediated by caspases and reactive oxygen species in LLC-PK1 cells.
Renal failure associated with aspergillosis is caused by pathogenic fungi. Gliotoxin is a toxic epipolythiodioxopiperazine metabolite produced by the pathogens. The present study investigated the cytotoxicity and underlying mechanisms induced by gliotoxin in LLC-PK1 cells, a porcine renal proximal tubular cell line. Gliotoxin at 100 ng/ml did not show a cytotoxic effect, but unmasked a dose-dependent cell death induced by TNF-alpha. TNF-alpha-induced cell death in the presence of gliotoxin was associated with hypodiploid nuclei and activation of caspase-3-like proteases. Blockade of caspases by boc-aspartyl (OMe)-fluoromethylketone and z-DEVD.fmk inhibited TNF-alpha-induced cell death. As the concentrations of gliotoxin were increased, gliotoxin killed the cells directly in a dose-dependent manner. Further analyses of DNA fragmentation, hypodiploid nuclei, mitochondrial membrane potential, and plasma membrane integrity revealed that cell death proceeded via apoptosis. Gliotoxin-induced apoptosis was associated with dose-dependent and time-dependent activation of caspase-3-like proteases. Boc-aspartyl (OMe)-fluoromethylketone attenuated the killing effect. Gliotoxin also increased the intracellular levels of reactive oxygen species as measured by flow cytometry. N-acetylcysteine, a well-known antioxidant, completely abolished the gliotoxin-induced caspase-3-like activity, cytotoxicity, and reactive oxygen species. In conclusion, (1) gliotoxin at 100 ng/ml unmasks the ability of TNF-alpha-induced apoptosis, and the effect of TNF-alpha is mediated by caspase-3-like proteases; and (2) at higher concentrations gliotoxin itself induces cell death, which is via apoptosis and dependent on caspase-3-like activity and reactive oxygen species. (+info)
Improved FACS analysis confirms generation of immature dendritic cells in long-term stromal-dependent spleen cultures.
Cells produced in spleen stroma-dependent long-term cultures (LTC) have now been clearly defined as dendritic cells (DC). Characterization of cells by antibody staining and FACS analysis has only been possible using a procedure to quench the high autofluorescence of DC produced in LTC (LTC-DC). The population of large cells produced by the established LTC-X1 culture are homogeneously positive for a number of cell-surface markers expressed by DC. These include CD11c, CD11b, Dec-205, Fc receptor and CD86. They also express markers detectable with the F4/80 and 33D1 antibodies. Cells produced in LTC do not uniformly express the MHC II marker, consistent with an immature DC phenotype. Most cells are weakly positive for MHC II with a small subset of highly positive cells. The quenching method involves staining cells with crystal violet dye, which is taken up within the cell. The importance of optimizimg fluorescent antibody staining assays for delineating DC subsets is indicated and the LTC system is established as a valuable and continuous source of DC precursors. (+info)
In vitro cytotoxicity of Acanthamoeba spp. isolated from contact lens containers in Korea by crystal violet staining and LDH release assay.
In order to observe the cytotoxicity of Acanthamoeba spp., which were isolated from contact lens containers as ethiological agents for the probable amoebic keratitis in Korea, the crystal violet staining method and LDH release assay were carried out. In the crystal violet staining method, among eight contact lens container isolates, isolate 3 (Acanthamoeba KA/LS5) showed 83.6% and 81.8% of cytotoxicity, and isolate 7 (Acanthamoeba KA/LS37) showed 28.2% and 25.1% of cytotoxicity, in 1 mg/ml and 0.5 mg/ml lysate treatments, respectively. Acanthamoeba culbertsoni and A. healyi showed 84.0% and 82.8% of cytotoxicity. Similar results were observed in A. castellanii and A. hatchetti which showed 83.6% and 75.5% of cytotoxicity. Acanthamoeba royreba and A. polyphaga showed 9.0% and 1.7% of cytotoxicity. In the LDH release assay, isolate 3 (20.4%) showed higher cytotoxicity than other isolates in 1 mg/ml lysate treatment. The results provide that at least isolate 3 has the cytotoxic effect against CHO cells and seems to be the pathogenic strain. (+info)
Prospective study of the usefulness of sputum Gram stain in the initial approach to community-acquired pneumonia requiring hospitalization.
From February 1995 through May 1997, we prospectively studied 533 patients with community-acquired pneumonia requiring hospitalization in order to assess the current usefulness of sputum Gram stain in guiding the etiologic diagnosis and initial antibiotic therapy when applied routinely. Sputum samples of good quality were obtained in 210 (39%) patients, 175 of whom showed a predominant morphotype. Sensitivity and specificity of Gram stain for the diagnosis of pneumococcal pneumonia were 57% and 97%, respectively; the corresponding values for Haemophilus influenzae pneumonia were 82% and 99%. Patients with a predominant morphotype were more frequently treated with monotherapy than were patients without a demonstrative sputum sample (89% vs. 75%; P<.001). Analysis of our data shows that a good-quality sputum sample can be obtained from a substantial number of patients with community-acquired pneumonia. Gram stain was highly specific for the diagnosis of pneumococcal and H. influenzae pneumonia and may be useful in guiding pathogen-oriented antimicrobial therapy. (+info)
Quantitative gram stain interpretation criteria used by microbiology laboratories in Alberta, Canada.
Microbiology laboratories in Alberta, Canada, were surveyed to determine the quantitative interpretive criteria used to routinely read and report Gram stains. There was a wide variability in the quantitative reporting criteria cited for both cells and bacteria, with only 11 of 32 (34.4%) laboratories surveyed using the criteria recommended by the external proficiency-testing program. Lack of standardized criteria not only poses a problem in the grading of proficiency testing results but may also impact the quality of patient care. (+info)